Klaus-Peter Wandinger

Association between clinical disease activity and Epstein–Barr virus reactivation in MS

Objective: To assess the potential significance of Epstein–Barr virus (EBV) reactivation in disease activity in MS patients. Methods: The prevalence of antibodies against herpes simplex virus type 1 (HSV-1), HSV-2, EBV, and cytomegalovirus was determined in a group of 108 MS patients and in 163 healthy control subjects. Sera were analyzed using combinations of novel assay systems employing highly purified viral and recombinant antigens. In addition, PCR for the detection of EBV DNA was performed in serial samples. Results: In contrast to the control populations, antibodies against EBV were present in 100% of MS patients. Among the tested human herpesviruses, this high extent of seropositivity was only found for EBV. Primary infection was found exclusively in the control group (3.7%), whereas serologic evidence of EBV reactivation was seen in MS patients (13.9%) as well as control subjects (17.2%). There was no temporal coincidence between EBV reactivation and disease activity in MS patients. However, in 19 patients followed monthly for 1 year, active viral replication as measured by increased immunoglobulin (Ig) M and IgA responses to EBV early antigens (p54 + p138) and positive serum DNA was seen in 72.7% of patients with exacerbations during the study period and in none of the patients with clinically stable disease. Conclusions: The results demonstrate an association between EBV reactivation and disease activity in MS patients over time, and suggest that EBV might play an indirect role in MS as an activator of the underlying disease process.

Increased Prevalence of Diverse N -Methyl-D-Aspartate Glutamate Receptor Antibodies in Patients With an Initial Diagnosis of Schizophrenia: Specific Relevance of IgG NR1a Antibodies for Distinction From N -Methyl-D-Aspartate Glutamate Receptor Encephalitis

CONTEXT Evidence for symptomatic convergence of schizophrenia and N-methyl-D-aspartate glutamate receptor (NMDA-R) encephalitis highlights the need for an assessment of antibody prevalence and specificity for distinct disease mechanisms in patients with a diagnosis of schizophrenia among glutamatergic pathophysiologic abnormalities in psychiatric disorders. OBJECTIVES To compare the specificity and prevalence of NMDA-R antibodies in schizophrenia (DSM-IV criteria) with those of other psychiatric diagnoses and to determine whether antibody subtypes characterize overlap with and distinction from those in NMDA-R encephalitis. DESIGN Serum from 459 patients admitted with acute schizophrenia, major depression (MD), and borderline personality disorder (BLPD) or individuals serving as matched controls was obtained from our scientific blood bank. To explore epitope specificity and antibody subtype, IgA/IgG/IgM NMDA-R (NR1a or NR1a/NR2b) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPA-R) (GluR1/GluR2) serum antibodies were determined. PARTICIPANTS Two hundred thirty matched healthy controls were compared with patients (unmedicated for at least 6 weeks) with schizophrenia (n = 121), MD (n = 70), or BLPD (n = 38). MAIN OUTCOME MEASURES The primary outcome was the overall number of seropositive cases for NMDA-R and AMPA-R antibodies; the secondary outcome was disease specificity of IgA/IgG/IgM antibodies and epitope specificity for clinical subgroups. RESULTS Diverse NMDA-R antibodies were identified in 15 subjects, primarily those with an initial schizophrenia diagnosis (9.9%), opposed to MD (2.8%), BLPD (0), and controls (0.4%). Retrospectively, 2 patients initially classified as having catatonic or disorganized schizophrenia were reclassified as having misdiagnosed NMDA-R encephalitis (presence of specific serum and cerebrospinal fluid IgG NR1a antibodies). In all other seropositive cases, the antibodies consisted of classes IgA and/or IgM or were directed against NR1a/NR2b (not against NR1a alone). None of the patients or controls had antibodies against AMPA-R. CONCLUSIONS Acutely ill patients with an initial schizophrenia diagnosis show an increased prevalence of NMDA-R antibodies. The repertoire of antibody subtypes in schizophrenia and MD is different from that with NMDA-R encephalitis. The latter disorder should be considered as a differential diagnosis, particularly in young females with acute disorganized behavior or catatonia.

Anti-NMDA-receptor encephalitis: A severe, multistage, treatable disorder presenting with psychosis

Anti-NMDA-receptor encephalitis is a severe, treatable and potentially reversible disorder presenting with memory deficits, psychiatric symptoms and seizures. Initially described in young patients with ovarian teratoma, the disease is meanwhile increasingly recognized also in women without tumours, in men and in children. The presence of anti-glutamate receptor (type NMDA) autoantibodies in serum or cerebrospinal fluid is specific for this novel and widely underdiagnosed disorder. Early recognition is crucial since prognosis largely depends on adequate immunotherapy and, in paraneoplastic cases, complete tumour removal. Indirect immunofluorescence using NMDA-type glutamate receptors recombinantly expressed in human cells is a highly competent method for diagnosing anti-NMDA-receptor encephalitis.

Cerebrospinal fluid findings in aquaporin-4 antibody positive neuromyelitis optica: results from 211 lumbar punctures.

BACKGROUND Neuromyelitis optica (NMO, Devic disease) is a severely disabling autoimmune disorder of the CNS, which was considered a subtype of multiple sclerosis (MS) for many decades. Recently, however, highly specific serum autoantibodies (termed NMO-IgG or AQP4-Ab) have been discovered in a subset (60-80%) of patients with NMO. These antibodies were subsequently shown to be directly involved in the pathogenesis of the condition. AQP4-Ab positive NMO is now considered an immunopathogenetically distinct disease in its own right. However, to date little is known about the cerebrospinal fluid (CSF) in AQP4-Ab positive NMO. OBJECTIVE To describe systematically the CSF profile of AQP4-Ab positive patients with NMO or its formes frustes, longitudinally extensive myelitis and optic neuritis. MATERIAL AND METHODS Cytological and protein biochemical results from 211 lumbar punctures in 89 AQP4-Ab positive patients of mostly Caucasian origin with neuromyelitis optica spectrum disorders (NMOSD) were analysed retrospectively. RESULTS CSF-restricted oligoclonal IgG bands, a hallmark of MS, were absent in most patients. If present, intrathecal IgG (and, more rarely, IgM) synthesis was low, transient, and, importantly, restricted to acute relapses. CSF pleocytosis was present in around 50% of samples, was mainly mild (median, 19 cells/μl; range 6-380), and frequently included neutrophils, eosinophils, activated lymphocytes, and/or plasma cells. Albumin CSF/serum ratios, total protein and CSF L-lactate levels correlated significantly with disease activity as well as with the length of the spinal cord lesions in patients with acute myelitis. CSF findings differed significantly between patients with acute myelitis and patients with acute optic neuritis at the time of LP. Pleocytosis and blood CSF barrier dysfunction were also present during remission in some patients, possibly indicating sustained subclinical disease activity. CONCLUSION AQP4-Ab positive NMOSD is characterized by CSF features that are distinct from those in MS. Our findings are important for the differential diagnosis of MS and NMOSD and add to our understanding of the immunopathogenesis of this devastating condition.

Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen

BACKGROUND Recently, a highly specific serum autoantibody was discovered in patients with neuromyelitis optica, called NMO-IgG, and aquaporin-4, the most abundant water channel in the CNS, was identified as the target antigen. Several assays for the detection of NMO-IgG/AQP4-Ab have been described. Tests based on recombinant human AQP4 have been repeatedly demonstrated to be more sensitive than the previous gold standard assay, i.e. immunohistochemistry (IHC) on mouse brain tissue. However, the sophisticated techniques applied restrict their availability to few laboratories worldwide. OBJECTIVE To develop an easy-to-use, recombinant immunofluorescence assay (rIFA) suitable for standardized and high-throughput detection of NMO-IgG/AQP4-Ab. METHODS HEK293 cells seeded on cover glasses were transfected with full-length recombinant human AQP4 at large scale. Cover glasses with the immobilized cells were cut into millimetre-sized fragments and transferred to microscopy slides. 151 serum samples from patients with NMO spectrum disorders (NMOSD) and controls were analysed both in the standard IHC assay and in the newly developed rIFA. RESULTS 25/32 (78.1%) patients with clinically definite NMO and 36/51 (70.6%) of total patients with NMOSD were positive for NMO-IgG/AQP4-Ab in the rIFA compared to 65.6% and 58.8%, respectively, in the IHC assay. CONCLUSION The recombinant IFA presented here provides laboratories familiar with indirect immunofluorescence microscopy with a highly sensitive and reproducible diagnostic tool for standardized detection of antibodies to AQP4. This new approach could make AQP4-Ab testing, which is of high clinical relevance, more widely available.

Cognitive deficits following anti-NMDA receptor encephalitis

Background Anti-NMDA receptor (NMDAR) encephalitis is a recently characterised autoimmune disorder mainly affecting young women. Although the clinical features of the acute disease are well characterised, cognitive long-term outcome has not been examined in detail. Methods The authors investigated cognitive performance in nine patients with proven anti-NMDAR encephalitis after recovery from the acute disease period (median 43 months after disease onset, range 23 to 69). Patients underwent a comprehensive neuropsychological assessment, including memory tasks that have previously been shown to be sensitive for hippocampal dysfunction. Results Substantial persistent cognitive impairments were observed in eight out of nine patients that mainly consisted of deficits in executive functions and memory. The severity of these deficits varied inter-individually. Patients with early immunotherapy performed significantly better. The most severe deficits were observed with inefficient or delayed initial treatment. Conclusion Our results suggest that cognitive deficits constitute a major long-term morbidity of anti-NMDAR encephalitis. These deficits relate to the distribution of NMDARs in the human brain and their functional role in normal cognition. Good cognitive long-term outcome may depend on early and aggressive treatment.

Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance

BackgroundIn 70-80% of cases, neuromyelitis optica (NMO) is associated with highly specific serum auto-antibodies to aquaporin-4 (termed AQP4-Ab or NMO-IgG). Recent evidence strongly suggests that AQP4-Ab are directly involved in the immunopathogenesis of NMO.ObjectiveTo assess the frequency, syndrome specificity, diagnostic relevance, and origin of cerebrospinal fluid (CSF) AQP4-Ab in patients with NMO spectrum disorders (NMOSD).Methods87 CSF samples from 37 patients with NMOSD and 42 controls with other neurological diseases were tested for AQP4-Ab in a cell based assay using recombinant human AQP4. Twenty-three paired CSF and serum samples from AQP4-Ab seropositive NMOSD patients were further analysed for intrathecal IgG synthesis to AQP4.ResultsAQP4-Ab were detectable in 68% of CSF samples from AQP4-Ab seropositive patients with NMOSD, but in none of the CSF samples from AQP4-Ab seronegative patients with NMOSD and in none of the control samples. Acute disease relapse within 30 days prior to lumbar puncture, AQP4-Ab serum titres >1:250, and blood-CSF barrier dysfunction, but not treatment status, predicted CSF AQP4-Ab positivity. A positive AQP4-specific antibody index was present in 1/23 samples analysed.ConclusionsAQP4-Ab are detectable in the CSF of most patients with NMOSD, mainly during relapse, and are highly specific for this condition. In the cohort analysed in this study, testing for CSF AQP4-Ab did not improve the sensitivity and specificity of the current diagnostic criteria for NMO. The substantial lack of intrathecal AQP4-Ab synthesis in patients with NMOSD may reflect the unique localisation of the target antigen at the blood brain barrier, and is important for our understanding of the immunopathogenesis of the disease.

Polyspecific, antiviral immune response distinguishes multiple sclerosis and neuromyelitis optica

Background: A polyspecific, intrathecal humoral immune response against neurotropic viruses such as measles, rubella and varicella zoster virus (MRZ reaction, MRZR) is present in 80–100% of patients with multiple sclerosis (MS), but has not to date been evaluated in patients with neuromyelitis optica (NMO). Aims: To evaluate whether MRZR distinguishes NMO and MS. Methods: 20 patients with NMO and 42 with MS were included. The intrathecal synthesis of antibodies against measles, rubella and varicella zoster virus was detected by calculation of the respective antibody indices (AI). Results: A positive MRZ reaction, as defined by a combination of at least two positive AIs, was found in 37/42 MS, but in only 1/20 NMO patients (p<0.0001). Median AI values differed significantly between the groups (p<0.0005). Conclusions: The polyspecific antiviral humoral immune response characteristic for MS is widely missing in NMO, irrespective of the NMO-IgG status of the patients. Our findings further strengthen the case for NMO being pathologically distinct from MS.

Neuromyelitis optica spectrum disorders in patients with myasthenia gravis: ten new aquaporin-4 antibody positive cases and a review of the literature.

Background: Neuromyelitis optica (NMO, Devic syndrome) and myasthenia gravis (MG) are rare antibody-mediated autoimmune disorders. Concurrent incidence has been reported in only few patients, mostly non-Caucasians. Objective: To report on ten Caucasian patients with NMO spectrum disorders (NMOSD) and MG and to provide a comprehensive review of the literature. Method: Retrospective study. Results: In total, 26 patients (m:f = 1:12; Caucasian in 12) with MG (generalized in 17) and NMOSD (NMO in 21, longitudinally extensive transverse myelitis in five) were identified from the authors’ own files (n = 10) and the previous literature (n = 16). MG preceded NMOSD in 24/25 cases (96%). AQP4-Ab were tested in 20 patients and were positive in 17 (85%). Twenty out of 25 patients (80%) had been treated with thymectomy or thymic irradiation, which preceded NMOSD in all cases (median latency, 12 years; range, 0.3–32). At last follow-up, complete remission of MG was reported in 15/22 (68%), and MG was well controlled with pyridostigmine in three. Co-existing autoimmune disorders or autoimmune antibodies were reported in 17 patients. Conclusion: Our study demonstrates that i) AQP4-Ab-positive NMOSD are more commonly associated with MG in Caucasians than previously thought; ii) MG precedes NMOSD in most cases, often by more than a decade; iii) NMOSD almost exclusively occur in females with juvenile or early-onset MG; and iv) MG frequently takes an unusually mild course in patients with NMOSD. A history of thymectomy could be a possible risk factor for the later development of NMOSD. We recommend testing for AQP4-Ab in MG patients presenting with atypical motor or optic symptoms.

Autoantibodies against aquaporin-4 in patients with neuropsychiatric systemic lupus erythematosus and primary Sjögren's syndrome.

Neurologic manifestations occur in up to 70% of patients with systemic lupus erythematosus (SLE) and in 20% of patients with Sjögren’s syndrome (SS) and are associated with significant morbidity. There is as yet no biologic marker that specifically indicates neurologic involvement in rheumatic diseases. In the literature to date, the association between nervous system manifestations of SLE and autoantibodies against ribosomal P proteins and N-methyl-Daspartate receptor has been inconsistently demonstrated (1). Transverse myelopathy is a rare but serious condition reported in patients with SLE and SS (2,3). In sera from some of these patients, an IgG autoantibody marker, called NMOIgG, has been recently detected (4,5). NMO-IgG was initially identified by immunohistochemistry using brain tissue in sera from patients with neuromyelitis optica (NMO; also known as Devic’s disease), a severe demyelinating disorder of the central nervous system (CNS) that primarily affects the spinal cord and the optic nerves (6). In patients presenting with isolated longitudinally extensive transverse myelitis (LETM) involving at least 3 vertebral segments, as revealed using magnetic resonance imaging, and in patients with recurrent optic neuritis (ON), the presence of NMO-IgG indicates severe disease course with frequent relapses (7,8). NMO-IgG is not detectable in the sera of patients with multiple sclerosis (MS) (6). The antigenic target of NMO-IgG is aquaporin-4 (AQP-4), the most abundant water channel in the CNS (9). To detect these autoantibodies, assays employing recombinant AQP-4 have been repeatedly shown to be more sensitive than immunohistochemistry in CNS tissue (10). In order to evaluate the significance of NMO-IgG/ AQP-4 antibodies as potential markers of neurologic involvement in rheumatic conditions, we collected serum samples from patients with SLE (n 48) and SS (n 44), 28 and 22 of whom showed neurologic manifestations, respectively (Table 1). All patients fulfilled the American College of Rheumatology (ACR) classification criteria for SLE and SS (11,12). SLE patients with neurologic involvement fulfilled the ACR case definitions for neuropsychiatric lupus syndromes (13). Blood was drawn from 3 SLE patients (2 with LETM and 1 with LETM and recurrent ON) within 7 days of onset of a relapse. In the other SLE subsets, the time intervals between the serum sampling and the onset of the first neurologic symptoms or, in cases of relapses, the onset of the last relapse were as follows: TM, 1 year; LETM, 1–7 years (median 4 years); recurrent ON, 1 year; chorea, 1 year; psychosis/depression, 4–20 years (median 9.5 years); seizure disorders, 13 and 20 years. The duration of symptoms in SLE patients with polyneuropathy was 2–12 years (median 5 years). In patients with SS, time intervals were 7 and 8 years in 2 patients with TM and concomitant monophasic ON, 5 years in 1 patient with TM alone, and 13 years in both patients with LETM. Symptoms were present for 1–8 years (median 3 years) in SS patients with polyneuropathy. Samples were analyzed both by immunohistochemistry using native primate cerebellum, cerebrum, and optic nerve tissue sections and, for the first time in patients with SLE and SS, by means of a recombinant immunofluorescence assay (rIFA) using AQP-4–transfected HEK cells fixed in formalin. In direct comparison with the original procedure for the detection of NMO-IgG as described by Lennon et al (6), the rIFA exhibited an increase in sensitivity of 12.5% (overall sensitivity 78.1%; specificity 100%) in an earlier study based on 183 samples from NMO patients and relevant neurologic disease controls, including MS (14). Serum samples were classified as being positive or negative for NMO-IgG/AQP-4 antibodies by 2 independent investigators who were unaware of the clinical data. Testing was performed for diagnostic purposes in all cases. In SLE and SS, NMO-IgG/AQP-4 antibodies were found exclusively in patients with neurologic involvement comprising LETM or recurrent ON (Table 1). In the SLE cohort, autoantibodies against AQP-4 were detected in all of these patients, whereas NMO-IgG was found in 6 of 7 individuals, once more indicating a higher sensitivity of the recombinant cell substrate compared with immunohistochemistry. Antibody titers as determined by AQP-4–transfected HEK cells ranged from 1:10–1:1,000 in the 5 patients with LETM. The titer was 1:100 in the patient with recurrent ON and 1:3,200 in the patient with LETM and recurrent ON. In the SS group, 1 of the 2 patients with LETM was positive for NMO-IgG/AQP-4 antibodies, at a titer of 1:1,000. None of the SLE and SS patients without neurologic involvement or symptoms other than LETM or recurrent ON tested positive for NMO-IgG/AQP-4 antibodies (i.e., at a starting dilution of 1:10, antibodies were not detected in the serum samples from these patients). Our data demonstrate that autoantibodies against AQP-4 generally are not a marker for neurologic involvement in SLE and SS. Consistent with the findings of previous reports, our observations strongly support the notion that the presence of autoantibodies against AQP-4 is highly correlated with a distinct clinical phenotype, i.e., LETM and recurrent ON, which together are typical for NMO (6–8). Apparently, autoantibodies against AQP-4 are a marker for NMO that may occur either as an isolated syndrome or as part of a rheumatologic disease like SLE or SS. Of particular interest is the fact that 2 of the AQP-4–positive SLE patients (1 with LETM and 1 with recurrent ON) also had myasthenia gravis. This observation further supports the concept of coexisting independent, antibody-mediated autoimmune disorders in the same patient. It can be argued that the long intervals between the onset of neurologic symptoms and the time of blood sampling in most of the patients might have influenced the serologic findings. However, it is known from NMO patients with long-term followup that AQP-4 antibodies are detectable in the serum during relapse as well as during remission, suggesting that AQP-4 antibody testing can be of diagnostic relevance independently of disease activity (15). In clinical practice, myelitis and ON occurring in patients with SLE and SS can be